Summary: Daily exposure to
fuel vapour may pose significant health risk to exposed individuals. Fifteen
each of male and female albino rats weighing between 110-230g were divided into
test (10) and control (5) groups each. The test animals were exposed to
inhalation gasoline for one hour daily for twenty-one consecutive days. All
animals were then bled and the serum levels of the reproductive hormones
determined. The results showed significant (P<0.05) reduction in the serum
levels of all the hormones of reproduction in both the male and female test
animals. The results suggest that inhalation gasoline exposure significantly
(P<0.05) lowers the levels of reproductive hormones in albino rats and may
thus interfere with reproduction.

Key Words: Gasoline, Wistar, hormones, reproduction.

Introduction

Endocrine disrupting compounds come from different sources. Gasoline, popularly
known as fuel, is a volatile, highly flammable liquid mixture of hydrocarbons. It
is used in internal combustion engines. Gasoline gains entrance into the body
orally (accidental), transdermally, and by inhalation. The inhalation route
would appear to be more important because more persons may be affected and
most exposed persons in Nigeria for instance are unprotected. Gasoline is a
mixture of over five hundred hydrocarbons with varied composition according
to its place of origin. In general, it contains alkanes, alkenes, alcohol,
ether and many additives such as benzene and lead (Isabelle et al, 1997). Toxic
effects of gasoline and additives range from induction of cancer to disruption
of haematopoiesis and endocrine functions (Schlosser et al, 1993 and
Orzechowski et al, 1995). The exposure levels of ethanol (an additive
of gasoline) in humans are thought to be lower than levels known to cause toxic
effects (Lovei, 1999). In developing countries such as Nigeria where oil exploration
is massive, occupational exposure may pose some amount of health risks. Some
specific endocrine endpoints are predictive of conceptive female sexual cycle
and male fertility status and can be used as early markers of possible reproductive
dysfunctions of low dose exposures to gasoline vapour. It is difficult to fully
assess the health risk posed to the many fuel pump attendants, refinery and
gasoline depots workers by prolonged exposure to inhalation gasoline, which
generally saturate the ambient air of their workplaces.

In this study, we challenged male and female albino rats with gasoline vapour
and monitored the endocrine disruptive effects as part of a comprehensive study
of the health risks faced by refinery workers in Nigeria. The ultimate goal
of the study included a definition of the potential impact of inhalation gasoline
on reproductive and endocrine functions of the industry workers. Much information
on the toxicological effects of gasoline vapour on humans in the country is
relatively scanty. However, casual relationships have been variously made
between gasoline inhalation and respiratory and central nervous system depression
and death (Steffe et al, 1960, renal cancer, liver toxicity and risk
of developing leukemia and myeloma (Enterline, 1993, Orzechowski et al, 1995)
in many other countries.

Materials and Methods

Animals:

Inbred male and female albino rats (wistar) aged between 15-20 weeks weighing
between 110g-130g were conditioned for this study in our laboratory. The animals
were exposed to 12:12 hours of light/dark periods and allowed free access to
water and their accustomed diet of grower’s mash (Guinea feeds, Nigeria). The
animals were randomized into four groups (A-D) of male or female rats each
as follows: Group A (female test group n = 10), Group B (male test group n
= 10), Group C (female control group n = 5), and Group D (male control group
n = 5). All the female rats were confirmed to be cycling normally and to be
sexually mature. This study was approved by the Research Ethics Committee
of the Nnamdi Azikiwe University Medical School. The animal care guidelines
of European Centre for the Validation of Alternative Methods (ECVAM, 1993)
were followed.

Gasoline Challenge:

The members of Groups A and B consisting of 10 females and 10 males were exposed
to gasoline vapour for 1 hour daily for 21 consecutive days. Exposure was
achieved by soaking 5ml of commercially procured gasoline in 20g of cotton
wool, which were plastered on one end of the rate cages. The vapour was allowed
to mix with the ambient air of the cages and the larger environment and about
1-2ml of the gasoline was vapourized in the ambient air of each rat cage for
the one hour. The exposure modality simulated a general occupational oil depot
environment in which gasoline and other derivatives saturated the ambient air
to which unprotected oil workers in Nigeria, are exposed daily for hours.

Monitoring the Estrus Cycling

The estrus cycling of the female test and control groups was monitored beginning
from the Day-1 of exposure. Estrus cycling was determined by the vaginal swab
method. The vaginal smears were stained with Leishmann’s stain and examined
under a light microscope.

Hormonal Assay

Serum samples were taken from all the animals (Groups A – D) after the 21
– day challenge. All samples were stored at zero degrees centigrade and analyzed
within 24 hours after collection. The levels of LH, FSH, Progesterone, Estradiol
and Testosterone were determined in the female and male animals. The determination
was made by the microwell enzyme-linked assay method using respective hormone’s
kit (Syntron Bioresearch Inc. USA).

Results

Effect of Gasoline Inhalation on Estrus Cycling.

The cycle of the female test animals (Group A) was categorized into estrus
weeks (Week I to V) covering the 21 days of challenge. The results show that
by the third estrus week, only 6 of the 10 individuals in the female test group
were still cycling and by the IV and V weeks, all the female rats exposed to
gasoline had persistent diestrous indicating arrest of estrus cycling by gasoline
inhalation. The results are summarized in table
1.

Hormonal Assay

The results of the hormonal assay of the male and female animals are summarized
in table II. The hormonal profile of the male test group show a significant
(P<0.05) decrease of the serum levels of testosterone at a mean of 2.7± 0.2
ng/ml relative to a mean level of 4.4 ± 0.7ng/ml for the male control group. On
the other hand, the levels of LH and FSH in the male test group were significantly
higher than the levels of the male control group. Gasoline inhalation appears
to significantly, cause a rise in the levels of LH and FSH in the male rats
as shown in table 2.

The hormonal profiles in the female test group show significant (P<0.05)
suppression in the levels of Progesterone and Estradiol (16±2.0 vs. 31± 18
vs 610 ± 40 ng/ml) respectively. However, whereas the level of FSH in the
female test group was higher relative to the control animals, the level of
LH in the test group was significantly (P>0.05) lower than that of the control
animals with means of 3.5 ± 0.5 vs. 6.5 ± 0.9 miu/ml (table
3).

Discussions

The goal of this study is to determine the potential influence of inhalation
gasoline on endocrine and reproductive structures and functions. We had used
animal models as part of a preliminary risk-assesment of occupational exposure
to fuel vapour in the fast expanding Nigerian oil industry. Our earlier report
showed profound disruption of the histopathological organization of the reproductive
organs of albino rats after a moderate fuel vapour exposure (Ugwoke et al,
2004). The histopathological analyses of the reproductive organs of uterus,
fallopian tube and ovary showed mild disruption over this relatively short
exposure period, however these organs showed only xanthogranumatous depositions
while the testes showed mild spermatocytic arrest. In this study we report
significant alterations in the levels of the reproductive hormones of the animals. The
levels of estrogen, progesterone and testosterone in the female and male rats
were significantly (P>0.05) suppressed (tables
2 and 3).

This suppression was also indicated by the arrest of the estrous cycling of
the female rats at diestrus (table
1).

Hydrocarbons found in fuels and solvents are common in the oil industry environment
and the occupationally exposed persons continuously inhale these compounds. The
effect of vapourized fuel on the endocrine system is only poorly understood. The
pre-ovulatory LH level of female US Air force Personnel is reportedly lowered
by exposure to vapourized fuel (Susan et al, 2002).

Our present report suggests a possible endocrine disruptive effect by uncertain
compounds in fuel. The effects of fuel on the levels of these hormones in
the rats show significant suppression of the LH levels of the female test animals.
This low LH level may negatively affect other LH – dependent physiologic functions
including ovulation. On the other hand, the elevation of Estradiol and Progesterone
levels with a concomitant significant suppression of FSH in the female animals
is consistent with a compensatory response arising from reduced negative feedback
on the hypothalamus-pituitary levels by the lowered Estrogen and Progesterone
levels. This suggests that the effect of the fuel was at the level of the
gonads. Gonadal level influence is further suggested by the lowering of the
serum testosterone levels of male rats challenged with gasoline vapour. The
animals had a significantly (P<0.05) lower testosterone level relative to
their control counterparts (table
2). These abnormal hormonal levels suggest
a disruption of steroidogenesis function in rats exposed to gasoline vapour. Translation
research will be required to determine the reproduction health risks faced
by occupational exposure to gasoline. The future direction of this study would
be to assess adverse outcome on human subjects occupationally exposed to fuel
vapour. There are sex and species-specific differences in outcome of exposure
to fuel. It remains uncertain what compound(s) in gasoline caused the observed
effects. Further research would also be required to decipher what constituent(s)
may be responsible for the observed effects.

Acknowledgements

We wish to thank the entire staff and management of Nnamdi Azikiwe University
and Ambiln Laboratory Enugu for making this research possible.

References

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